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J. Tsai, R.N. Fariss, T. Yamamoto, F.I. Hickman; A Novel Transgenic Mouse Strain Expressing Green Fluorescent Protein (GFP) in Retinal Pigment Epithelial (RPE) Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2288.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously generated transgenic mice (SMAA-GFP) expressing GFP under control of the smooth muscle alpha actin (SMAA) promoter. Most of SMAA-GFP mice express GFP in vascular smooth muscle cells and some pericytes. Here we report that one line of these mice, E7, exhibits intense GFP expression throughout the entire retinal pigment epithelium. Methods: RPE cells were identified by immunostaining with rabbit anti-RPE-65 antibodies (Dr. Michael Redmond, NEI) and an Alexa568-conjugated goat anti-rabbit secondary antibody. To culture E7 RPE cells, the posterior eyecups were incubated in 2% dispase (Life Technologies) at 37 OC for 50 minutes. Sheets of RPE cells were removed and plated on laminin-coated plates in DMEM/F12 medium plus 10% FBS. For short interference RNA (siRNA) transfection, the third passaged E7 RPE primary cells were transfected with siRNA duplex using TransMessenger transfection reagent (Qiagen) with siRNA duplexes for GFP and control(Xeragon). Results: Examination of longitudinal sections of the retina and flat mount of posterior eyecups revealed that both nuclei and cytoplasm of RPE cells from E7 mice are brightly GFP fluorescent. The cytoplasmic GFP signal was completely colocalized with the RPE cell-specific marker RPE65. The isolated RPE cells continued expressing high levels of GFP in culture, which can be suppressed by siRNAs duplex against GFP. Conclusions: Unlike other SMAA-GFP mice, the RPE cells of E7 mice are intensely labeled with GFP. The position of the transgene insertion is most likely responsible for this RPE expression. The analysis of the sequences flanking the transgene insertion site will thus provide new insights into RPE cell-specific gene expression. In addition, E7 mice are a valuable source of GFP positive RPE cells for RPE cells transplantation studies as well as a useful model to evaluate siRNA based therapeutic strategies targeting RPE cells.
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